【摘要】：食管癌(Esophageal carcinoma,EC)是一種最常見的消化道惡性腫瘤,位居全世界癌癥發(fā)病率第四位。在中國食管癌已經(jīng)成為主要的消化道腫瘤死因之一�，F(xiàn)階段尚不清楚食管癌的確切病因,臨床上大多數(shù)食管癌患者就診時已處于中晚期,近年來隨著外科診療水平的提高其術(shù)后5年生存率僅僅為25%左右。然而,早期食管癌術(shù)后5年生存率可達(dá)90%以上。為此探討針對食管癌的早期致病機(jī)制,已經(jīng)成為臨床食管癌主要的研究方向之一。目前為止,食管癌致病機(jī)制的臨床和基礎(chǔ)研究仍無突破。隨著分子生物學(xué)技術(shù)研究的提高,已經(jīng)證實在頭頸部鱗癌、肺癌、口腔癌、結(jié)腸癌中包括食管鱗癌在內(nèi)的多種上皮來源性的腫瘤中都有可能存在“區(qū)域癌化”等早期癌變現(xiàn)象。水通道蛋白-3(AQP-3,aquaporin-3)作為水通道蛋白家族中一重要亞型,具有該蛋白家族基因的共同特性,研究發(fā)現(xiàn)AQP3曾作為致癌基因在食管癌的分化增殖和侵襲轉(zhuǎn)移中起著重要的作用。目前對于食管鱗癌的研究國內(nèi)很少見從“區(qū)域癌化”理論角度來探討食管癌早期發(fā)病機(jī)制,尤其缺乏對食管鱗癌區(qū)域癌化內(nèi)水通道蛋白-3(AQP3)表達(dá)異常的系統(tǒng)性認(rèn)識及相關(guān)臨床研究。近年研究結(jié)果顯示,AQP3在多種不同惡性腫瘤內(nèi)表達(dá)水平與腫瘤的增殖分化、遠(yuǎn)處侵襲轉(zhuǎn)移及生存預(yù)后存在相關(guān)性。依據(jù)現(xiàn)有文獻(xiàn)報道,在食管鱗癌不同組織區(qū)域中存在AQP3表達(dá)差異,其中AQP3表達(dá)含量與食管鱗癌的臨床預(yù)后具有相關(guān)性。本研究采用Lugol's染色及HE化學(xué)染色法初步篩選出食管鱗癌中具有“區(qū)域癌化組織”的標(biāo)本,通過Real-Time PCR實時熒光定量和Western-blot蛋白質(zhì)免疫印跡技術(shù)從基因和蛋白質(zhì)水平驗證AQP3基因在食管正常組織、區(qū)域癌化及配對鱗癌組織中的表達(dá)差異,并分析其與相關(guān)影響因素之間的關(guān)系;結(jié)合術(shù)后隨訪系統(tǒng)評估食管鱗癌患者AQP3表達(dá)水平及相關(guān)臨床預(yù)后因素對生存時間的影響;最后從基因、蛋白質(zhì)組學(xué)方面為食管鱗癌的早期發(fā)病機(jī)制提供新的思路。目的:依據(jù)食管鱗癌“區(qū)域癌化”理論基礎(chǔ),用Lugol's染色及HE化學(xué)染色法初步篩選出食管鱗癌中“區(qū)域癌化組織”即Lugol's液染色陽性病理表型正常的食管粘膜組織。利用Real-Time PCR實時熒光定量和Western-blot蛋白質(zhì)免疫印跡技術(shù)驗證水通道蛋白-3(AQP3)在食管正常上皮、區(qū)域癌化及食管鱗癌三種不同組織中的表達(dá),并結(jié)合臨床相關(guān)預(yù)后因素探討AQP3表達(dá)與食管鱗癌生存時間的關(guān)系,初步了解AQP3基因在食管鱗癌癌變過程中的作用。方法:(1)回顧性分析2005年1月至2015年12月間甘肅省人民醫(yī)院及甘肅省腫瘤醫(yī)院胸外科食管鱗癌手術(shù)標(biāo)本共102例。所有收集的標(biāo)本均行食管粘膜Lugos’s液染色,初步篩選出65例患者存在“區(qū)域癌化”組織;(2)將65例符合納入標(biāo)準(zhǔn)的標(biāo)本分別采用Real-Time PCR和Western-blot從基因及蛋白層面檢測水通道蛋白-3(AQP3)在65例食管鱗癌區(qū)域癌化組織中的表達(dá)情況。(3)依據(jù)病歷查詢系統(tǒng)及生存期回訪系統(tǒng)回顧性收集65例患者臨床相關(guān)信息。采用Kaplan-Meier生存曲線分析比較各相關(guān)獨(dú)立因素生存期;采用Log-Rank檢驗比較各組間生存時間差異;Cox回歸模型分析判斷影響食管鱗癌預(yù)后的獨(dú)立因素,以α=0.05為差異有顯著統(tǒng)計學(xué)意義。結(jié)果1.Lugol's染色及HE化學(xué)染色法初步篩選出食管鱗癌田野癌化區(qū)域。證實了碘染色異常但病理表型正常的食管上皮符合區(qū)域癌化的條件,是食管鱗癌的早期病變。2.AQP3mRNA、蛋白表達(dá)水平與食管鱗癌的關(guān)系。AQP3mRNA、蛋白表達(dá)在三種不同食管組織中存在顯著的統(tǒng)計學(xué)差異,其表達(dá)含量依次升高(P0.05)。男性、家族史、淋巴結(jié)轉(zhuǎn)移、高中分化、浸潤深度、腫瘤大小,與AQP3mRNA陽高性表達(dá)率相關(guān),且AQP3表達(dá)蛋白的變化趨勢與AQP3基因表達(dá)具有一致性。3.生存分析Kaplan-Meier生存分析顯示:食管鱗癌生存期與性別、家族史、淋巴結(jié)轉(zhuǎn)移、浸潤深度、分化程度、腫瘤病理形態(tài)、AQP3mRNA及蛋白表達(dá)等單因素相關(guān)。Cox風(fēng)險回歸模型顯示:性別、淋巴結(jié)轉(zhuǎn)移、分化程度、浸潤深度及AQP3mRNA、蛋白陽性表達(dá)情況為食管鱗癌的獨(dú)立預(yù)后因素。結(jié)論總之本課題首次篩選出食管鱗癌病灶外區(qū)域癌化組織,然后從基因組、蛋白組等不同層面驗證了食管鱗癌區(qū)域癌化中差異基因AQP3的表達(dá)情況,并結(jié)合臨床病理指標(biāo)分析AQP3表達(dá)與食管鱗癌生存期之間的相關(guān)性,從而使人們對區(qū)域癌化的本質(zhì)有更深入的了解,為尋找食管鱗癌致病的上游靶點提供了新的研究方向。
[Abstract]:Esophageal carcinoma (EC) is one of the most common malignant tumors in the digestive tract, ranking fourth in the world. In China, esophageal cancer has become one of the main causes of death in the digestive tract. The exact cause of esophageal cancer is not clear at this stage. Most of the patients with esophageal cancer have been in the middle and late stages of clinic, and in recent years, the most of the patients are in the middle and late stages. With the improvement of surgical diagnosis, the survival rate of 5 years after surgery is only about 25%. However, the 5 year survival rate of early esophageal carcinoma can reach 90%. Therefore, the early pathogenesis of esophageal cancer has become one of the main research directions in the clinical esophagus cancer. So far, the clinical and basic Research of the pathogenesis of esophageal cancer is still still in the clinical and basic research. No breakthrough. With the improvement of molecular biological technology, it has been proved that early cancerous phenomena such as "regional carcinogenesis" are possible in the head and neck squamous cell carcinoma, lung cancer, oral cancer, and carcinoma of the esophagus including squamous carcinoma of the esophagus. The water channel protein -3 (AQP-3, aquaporin-3) is used as the aquaporin family. An important subtype, with the common characteristics of the protein family, has been found that AQP3 has played an important role in the proliferation and invasion and metastasis of esophageal cancer as a oncogene. At present, it is rare to explore the early pathogenesis of esophageal cancer from the perspective of "regional carcinogenicity", especially in the study of esophageal squamous cell carcinoma. Systematic understanding of abnormal expression of -3 (AQP3) in carcinogenesis of squamous cell carcinoma and related clinical studies. Recent results show that the expression level of AQP3 in a variety of different malignant tumors is related to tumor proliferation and differentiation, distant invasion and metastasis and survival prognosis. According to the existing literature, different tissues of esophageal squamous cell carcinoma are reported. There is a difference in the expression of AQP3 in the domain, in which the expression of AQP3 is correlated with the clinical prognosis of esophageal squamous cell carcinoma. In this study, the specimens with "regional cancerous tissue" in esophageal squamous cell carcinoma were screened by Lugol's staining and HE chemical staining, and the Real-Time PCR real-time fluorescence determination and Western-blot protein immunoblotting technique were used. Gene and protein levels were used to verify the difference in the expression of AQP3 gene in normal esophageal tissue, regional carcinogenesis and paired squamous cell carcinoma, and to analyze the relationship between the gene and related factors, and to evaluate the expression level of AQP3 in the patients with esophageal squamous cell carcinoma and the effect of related clinical prognostic factors on the survival time. The white matter group provides a new idea for the early pathogenesis of esophageal squamous cell carcinoma. Objective: Based on the theory of "regional carcinogenicity" of esophageal squamous cell carcinoma, Lugol's staining and HE chemical staining were used to preliminarily screen the esophageal mucosa tissues of "regional cancerous tissue" in esophageal squamous cell carcinoma, that is, the positive pathological phenotype of Lugol's liquid staining, and the use of Real-Time. PCR real-time fluorescence quantification and Western-blot protein immunoblotting were used to verify the expression of aquaporin -3 (AQP3) in three different tissues of normal esophageal epithelium, regional carcinogenesis and esophageal squamous cell carcinoma, and the relationship between the expression of AQP3 and the survival time of esophageal squamous cell carcinoma combined with clinical prognostic factors, and a preliminary understanding of the AQP3 gene in squamous carcinoma of the esophagus Methods: (1) retrospective analysis of 102 cases of esophageal squamous cell carcinoma in Gansu people's Hospital and Gansu cancer hospital from January 2005 to December 2015. All the collected specimens were stained with Lugos' s solution of esophageal mucosa, and 65 cases were preliminarily screened for "regional cancerous" tissue; (2) 65 cases were conformed to Nana. The standard specimens were used to detect the expression of aquaporin -3 (AQP3) in the cancerous tissues of 65 cases of esophageal squamous cell carcinoma using Real-Time PCR and Western-blot, respectively. (3) the clinical information of 65 patients was collected according to the medical record query system and the survival time return system. The Kaplan-Meier survival curve was used. The survival time of each related independent factor was compared and compared. Log-Rank test was used to compare the difference of survival time between each group; Cox regression model was used to determine the independent factors affecting the prognosis of esophageal squamous cell carcinoma, and the difference of alpha =0.05 was significant. Results 1.Lugol's staining and HE chemical staining were used to screen out the carcinogenesis area of esophageal squamous cell carcinoma. The esophageal epithelium with abnormal iodine staining but normal pathological phenotype accords with the condition of regional carcinogenesis. It is an early pathological change of esophageal squamous cell carcinoma (.2.AQP3mRNA). The relationship between protein expression level and esophageal squamous cell carcinoma is.AQP3mRNA. The protein expression in three different esophageal tissues has significant statistical difference, and the expression level of the esophageal squamous cell carcinoma is increased in turn (P0.05). History, lymph node metastasis, high school differentiation, depth of invasion, tumor size, correlation with AQP3mRNA Yang high expression rate, and the variation trend of AQP3 expression protein with AQP3 gene expression,.3. survival analysis Kaplan-Meier survival analysis showed that the survival period of esophageal squamous cell carcinoma and sex, family history, lymph node metastasis, depth, degree of differentiation, tumor A single factor related.Cox risk regression model, such as pathological morphology, AQP3mRNA and protein expression, showed that sex, lymph node metastasis, degree of differentiation, depth of infiltration and AQP3mRNA, and positive expression of protein were independent prognostic factors of esophageal squamous cell carcinoma. Conclusion in conclusion, this topic first screened out cancerous tissue in the outer region of the lesions of the squamous cell carcinoma of the tube, and then from the genome, The expression of differential gene AQP3 in the carcinogenesis of esophageal squamous cell carcinoma was verified at different levels, and the correlation between the expression of AQP3 and the survival period of esophageal squamous cell carcinoma was analyzed in combination with the clinicopathological indexes, thus making people more understanding of the nature of regional carcinogenesis, and providing a new research for the search of the upstream target of the esophageal squamous cell carcinoma. Look at the direction.
【學(xué)位授予單位】：甘肅中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.1
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本文編號：2166847